Analytical factors for optimization of chain-length distribution analysis of mRNA using capillary gel electrophoresis

• mRNA length is crucial in the quality assessment of mRNA therapeutics. • Capillary gel electrophoresis (CGE) is used to evaluate mRNA quality. • This study comprehensively analyzed the analytical parameters of CGE. • This study revealed the limitation of analyzing mRNA integrity. • Our analytical conditions showed higher RNA separation than those in USP. The development of mRNA vaccines against coronavirus disease-2019 has garnered attention in utilizing mRNA-based modalities such as mRNA vaccines and therapeutics. mRNA length is a crucial factor in the quality assessment of mRNA-based modalities, confirming the integrity of mRNA as an active component. Capillary gel electrophoresis is a representative analytical method used to examine mRNA chain length and should sufficiently separate full-length mRNAs and impurity RNAs (shortmers and longmers) to accurately evaluate mRNA quality. However, the analytical parameters affecting the separation of mRNAs have not been adequately verified. This study comprehensively analyzed the analytical parameters of capillary gel electrophoresis, and found that the gel concentration, denaturant, preheating treatment, capillary temperature, and fluorescent dye remarkably affect the separation of long-chain-length RNAs. The separation limits of capillary gel electrophoresis analysis were examined under adjusted analytical parameter conditions. Consequently, RNAs of approximately 4,000 nucleotides length and their defective RNAs of ≥200 nucleotides could be effectively separated. Furthermore, when RNA separation under our adjusted conditions was compared with that under the conditions recommended in the United States Pharmacopeia (USP) draft guidelines, our method demonstrated higher RNA separation for both RNA prepared in this study and that of the approved mRNA vaccine.